Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was conducted according to the appropriate OECD test guideline, and in compliance with GLP. The original study was considered reliability 1. Read-across to the registered substance is considered scientifically justified and is reliability 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2000

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

METI, MHLW and MAFF

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid

EC Number:

227-833-4

EC Name:

[[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid

Cas Number:

5995-42-6

Molecular formula:

C4H13NO7P2 (Constituent 1, linear form) C4H11NO6P2 (Constituent 2, cyclic form)

IUPAC Name:

[[(2-hydroxyethyl)imino]bis(methylene)]bisphosphonic acid

Test material form:

other: solution

Method

Target gene:

Histidine operon (Salmonella strains); tryptophan operon (E. coli)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbitone/beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

50-5000 µg active acid/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no information in study report

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Migrated to IUCLID6: 3 µg/plate TA 100, 5 µg/plate TA 1535, 2 µg/plate WP2uvrA

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation

Migrated to IUCLID6: 80 µg/plate TA 1537

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation

Migrated to IUCLID6: 2 µg/plate TA 98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 1 µg/plate TA 100, 2 µg/plate TA 1535 and TA 1537, 10 µg/plate E. coli WP2uvrA

Remarks:

with metabolic activation

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Migrated to IUCLID6: 5 µg/plate TA 98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours

SELECTION AGENT (mutation assays): histidine/tryptophan deficient agar

NUMBER OF REPLICATIONS: Plated in triplicate, experiment repeated


DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn

METABOLIC ACTIVATION: S9 mix included glucose-6-phosphate, NADP and NADPH as co-factors. 0.5 ml of 10% S9 solution was added to 0.1 ml test material, 0.1 ml of bacterial culture and 2 ml of top agar - final concentration approx 2% S9.

Evaluation criteria:

A test material is considered positive if it produces a reproducible, dose-related and statistically significant increase in the revertant count of at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No test-specific confounding factors were reported, and no precipitation or toxicity were observed.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Cytotoxicity assay, revertants per plate

+/- S9	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA 100	68	67	82	76	75	71	67	80	65	56	60
+		89	89	106	103	96	120	80	129	87	87	86*
-	WP” uvrA	20	24	22	15	20	16	20	22	17	28	23
+		24	21	30	37	30	30	21	13	41	28	39

* Sparse bacterial background lawn

Table 2 Experiment 1, plate incorporation, revertants per plate (mean of 3 plates)

Dose (µg/plate)	TA 100		TA1535		E. coli WP2 uvrA		TA 98		TA1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Negative control**	112	-	33	-	17	-	29	-	12	-
0*	99	102	33	16	29	23	20	33	13	17
50	93	99	33	16	21	24	14	30	11	16
150	96	94	41	17	23	25	21	23	14	14
500	114	97	28	15	22	25	20	34	12	15
1500	109	99	32	14	21	28	18	33	12	15
5000	95	75	31	16	24	26	19	24	12	15
Positive control	442	868	211	314	816	1002	166	27117	1177	466

* solvent control with water

** spontaneous reversion rate

Table 2 Experiment 2, plate incorporation, revertants per plate (mean of 3 plates)

Dose (µg/plate)	TA 100		TA1535		E. coli WP2 uvrA		TA 98		TA1537
	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Negative control**	120	-	27	-	25	-	25	-	12	-
0*	108	123	31	14	27	34	24	39	12	14
50	122	124	29	11	19	28	22	35	13	17
150	127	124	32	21	27	28	22	38	17	15
500	117	120	36	17	31	35	25	39	12	19
1500	120	121	34	19	27	31	17	31	15	14
5000	113	109	33	22	28	34	22	45	16	17
Positive control	363	1232	251		615	1146	138	247	982	330

* solvent control with water

** spontaneous reversion rate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

HEBMP-H has been tested for mutagenicity to bacteria in a reliable assay conducted according to OECD 471 and under GLP. No increase in the number of revertants per plate was found with and without activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and E.col WP2 uvrA in either the initial or the repeat plate incorporation assay. Appropriate positive, negative (spontaneous reversion) and solvent controls were included and gave expected results. It is concluded that the test substance is negative for the induction of mutation in bacteria under the conditions of the test.